Human glutaredoxin 3 forms [2Fe-2S]-bridged complexes with human BolA2

Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein, we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to those of the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans.     

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.     

Monothiol CGFS Glutaredoxins and BolA-like Proteins: [2Fe-2S] Binding Partners in Iron Homeostasis

Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S] bridging interaction between CGFS Grxs and a BolA-like protein in S. cerevisiae provided the first molecular-level understanding of the iron regulation mechanism in this model eukaryote and established the ubiquitous CGFS Grxs and BolA-like proteins as novel Fe-S cluster-binding regulatory partners. Parallel studies focused on Escherichia coli and human homologues for CGFS Grxs and BolA-like proteins have supported the studies in yeast and provided additional clues about their involvement in cellular iron metabolism. Herein, we review recent progress in uncovering the cellular and molecular mechanisms by which CGFS Grxs and BolA-like proteins help regulate iron metabolism in both eukaryotic and prokaryotic organisms.     

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1⁺ gene encoding a putative A-type Fe-S scaffold, in addition to mas5⁺ and hsc1⁺ genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2⁺, but not isu1⁺ and ssc1⁺, complemented the growth phenotype of Δgrx5 mutant as isa1⁺ did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.     

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.     

An Allelic Mutant Series of ATM3 Reveals Its Key Role in the Biogenesis of Cytosolic Iron-Sulfur Proteins in Arabidopsis

The ATP-binding cassette transporters of mitochondria (ATMs) are highly conserved proteins, but their function in plants is poorly defined. Arabidopsis (Arabidopsis thaliana) has three ATM genes, namely ATM1, ATM2, and ATM3. Using a collection of insertional mutants, we show that only ATM3 has an important function for plant growth. Additional atm3 alleles were identified among sirtinol-resistant lines, correlating with decreased activities of aldehyde oxidases, cytosolic enzymes that convert sirtinol into an auxin analog, and depend on iron-sulfur (Fe-S) and molybdenum cofactor (Moco) as prosthetic groups. In the sirtinol-resistant atm3-3 allele, the highly conserved arginine-612 is replaced by a lysine residue, the negative effect of which could be mimicked in the yeast Atm1p ortholog. Arabidopsis atm3 mutants displayed defects in root growth, chlorophyll content, and seedling establishment. Analyses of selected metal enzymes showed that the activity of cytosolic aconitase (Fe-S) was strongly decreased across the range of atm3 alleles, whereas mitochondrial and plastid Fe-S enzymes were unaffected. Nitrate reductase activity (Moco, heme) was decreased by 50% in the strong atm3 alleles, but catalase activity (heme) was similar to that of the wild type. Strikingly, in contrast to mutants in the yeast and mammalian orthologs, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. Our data suggest that Arabidopsis ATM3 may transport (1) at least two distinct compounds or (2) a single compound required for both Fe-S and Moco assembly machineries in the cytosol, but not iron.Plant cells contain more than 50 iron-sulfur (Fe-S) enzymes that carry out important redox and catalytic functions in many aspects of metabolism (Imsande, 1999; Balk and Lobréaux, 2005). The assembly of Fe-S cofactors is mediated by dedicated machinery of ancient evolutionary origin. In plants, mitochondria harbor homologs of the bacterial ISC (for iron sulfur cluster) proteins, while plastids have inherited the sulfur mobilization machinery from their cyanobacterial ancestor (Balk and Lobréaux, 2005; Kessler and Papenbrock, 2005; Pilon et al., 2006). Plant cytosol contains homologs of the cytosolic Fe-S assembly proteins that have recently been identified in yeast (Lill and Mühlenhoff, 2008), including the scaffold protein AtNBP35 (Bych et al., 2008; Kohbushi et al., 2009) and the hydrogenase-like AtNAR1 (Cavazza et al., 2008). It is thought that cytosolic Fe-S cluster assembly is dependent on at least one of the organelles, because the Cys desulfurases that generate sulfur for Fe-S clusters, CpNifS and NFS1, are strictly localized in the plastids and mitochondria, respectively (Kushnir et al., 2001; Frazzon et al., 2007; Van Hoewyk et al., 2007).In yeast, cytosolic and nuclear Fe-S cluster assembly depends on the mitochondrial ISC pathway and on the ATP-binding cassette (ABC) transporter of the mitochondria, Atm1p (Kispal et al., 1999). Atm1p is classified as a “half-transporter” that functions as a homodimer and is localized in the mitochondrial inner membrane with the ATPase domains at the matrix side (Leighton and Schatz, 1995). The orientation indicates that the direction of transport is from the mitochondrial matrix to the intermembrane space and cytosol. In accordance, mutations of yeast ATM1 cause a defect in cytosolic/nuclear Fe-S cluster assembly but not in mitochondrial Fe-S cluster assembly (Kispal et al., 1999). The substrates of Atm1p, however, or of its functional orthologs in other eukaryotes have not been identified thus far.Yeast atm1 mutations also disrupt iron homeostasis: iron uptake transporters are constitutively expressed independent of the iron concentration, and iron accumulates 10- to 30-fold in the mitochondria (Kispal et al., 1997, 1999). Mutations in the human ortholog ABCB7 are the cause of X-linked sideroblastic anemia with ataxia, in which one of the symptoms is mitochondrial iron overload (Rouault and Tong, 2008). Moreover, ATMs are widespread and highly conserved in β-proteobacteria and could be involved in nickel and cobalt resistance (Mikolay and Nies, 2009).The Arabidopsis (Arabidopsis thaliana) genome contains more than 100 ABC transporters, of which the ATMs form a small subfamily in the B group consisting of three genes: ATM1, ATM2, and ATM3 (Rea, 2007). The genes were first identified in Arabidopsis by Kushnir et al. (2001) and were named STA2, STA3, and STA1, respectively. (A proposal for a unified nomenclature [Verrier et al., 2008] has suggested the gene symbols AtABCB24, AtABCB23, and AtABCB25 for AtATM1, AtATM2, and AtATM3, respectively. However, for consistency with previous publications, we have adhered to the ATM gene symbols in this paper.) Expression of GFP fusions showed that all three ATM proteins localized to mitochondria (Kushnir et al., 2001; Chen et al., 2007). Arabidopsis ATM3 could functionally complement the yeast atm1 phenotype (Kushnir et al., 2001; Chen et al., 2007), whereas Arabidopsis ATM1 complemented poorly and ATM2 expression was toxic in yeast (Chen et al., 2007). Until now, functional analysis of the ATM genes in Arabidopsis has been restricted to one atm3 mutant, called starik (Kushnir et al., 2001), in which the protein lacks the C-terminal ATPase domain. The atm3-1 (sta1) mutant is dwarfed and chlorotic, but the biochemical basis of its pleiotropic phenotypes is unknown. In addition, induced expression of ATM3 in response to cadmium and lead, as well as sensitivity of the atm3-1 (sta1) mutant to cadmium, have implicated the ATM3 transporter in heavy metal tolerance (Kim et al., 2006).We have analyzed a comprehensive set of mutants in the Arabidopsis ATM genes and found that ATM3 plays a key role in plant metabolism, while mutants in ATM1 and ATM2 did not display an obvious phenotype. Genetic and biochemical evidence from an atm3 allelic series showed that ATM3 is important for the activity of cytosolic Fe-S and molybdenum cofactor (Moco) enzymes, but it does not play a major role in metal homeostasis.     

The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage

In photosynthetic eukaryotes assembly components of iron-sulfur (Fe-S) cofactors have been studied in plastids and mitochondria, but how cytosolic and nuclear Fe-S cluster proteins are assembled is not known. We have characterized a plant P loop NTPase with sequence similarity to Nbp35 of yeast and mammals, a protein of the cytosolic Cfd1-Nbp35 complex mediating Fe-S cluster assembly. Genome analysis revealed that NBP35 is conserved in the green lineage but that CFD1 is absent. Moreover, plant and algal NBP35 proteins lack the characteristic CXXC motif in the C terminus, thought to be required for Fe-S cluster binding. Nevertheless, chemical reconstitution and spectroscopy showed that Arabidopsis (At) NBP35 bound a [4Fe-4S] cluster in the C terminus as well as a stable [4Fe-4S] cluster in the N terminus. Holo-AtNBP35 was able to transfer an Fe-S cluster to an apoprotein in vitro. When expressed in yeast, AtNBP35 bound 55Fe dependent on the cysteine desulfurase Nfs1 and was able to partially rescue the growth of a cfd1 mutant but not of an nbp35 mutant. The AtNBP35 gene is constitutively expressed in planta, and its disruption was associated with an arrest of embryo development. These results show that despite considerable divergence from the yeast Cfd1-Nbp35 Fe-S scaffold complex, AtNBP35 has retained similar Fe-S cluster binding and transfer properties and performs an essential function.     

Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli

The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron‑sulfur clusters

Numerous proteins require iron?sulfur (Fe-S) clusters as cofactors for their function. Their biogenesis is a multi-step process occurring in the cytosol and mitochondria of all eukaryotes and additionally in plastids of photosynthetic eukaryotes. A basic model of Fe-S protein maturation in mitochondria has been obtained based on studies achieved in mammals and yeast, yet some molecular details, especially of the late steps, still require investigation. In particular, the late-acting biogenesis factors in plant mitochondria are poorly understood. In this study, we expressed the factors belonging to NFU, BOLA, SUFA/ISCA and IBA57 families in the respective yeast mutant strains. Expression of the Arabidopsis mitochondrial orthologs was usually sufficient to rescue the growth defects observed on specific media and/or to restore the abundance or activity of the defective Fe-S or lipoic acid-dependent enzymes. These data demonstrate that the plant mitochondrial counterparts, including duplicated isoforms, likely retained their ancestral functions. In contrast, the SUFA1 and IBA57.2 plastidial isoforms cannot rescue the lysine and glutamate auxotrophies of the respective isa1-isa2Δ and iba57Δ strains or of the isa1-isa2-iba57Δ triple mutant when expressed in combination. This suggests a specialization of the yeast mitochondrial and plant plastidial factors in these late steps of Fe-S protein biogenesis, possibly reflecting substrate-specific interactions in these different compartments.